A. PROJECT SUMMARY Nuclear bodies (NBs) are ubiquitous membrane-less structures that play important but poorly-understood roles in gene regulation. NBs locally increase the concentration of molecules involved in chromatin remodeling, transcription initiation, and RNA processing. Despite their functional importance, and decades of study, we lack a quantitative, mechanistic understanding of NB assembly. Understanding the biophysical rules governing NB assembly and properties is key to elucidating their function. Our group has pioneered the concept that NBs are liquid phase droplets that assemble through phase transitions. Here we will build on this framework, and test it, by developing a new technology that uses light to control nucleoplasmic phase transitions. This technology will enable precise spatiotemporal control of the assembly of NBs and their viscoelastic properties, as well as testing the impact on composition, function, and genome architecture. Our team is uniquely positioned to develop this exciting technology and exploit it to studying NBs, both in these Specific Aims, as well as together in future collaborations within the 4D Nucleome Program. Figure1. Schematic showing light- responsive droplets for interrogating NB assembly, properties, and function. AIM 1. Build and Characterize Purified Optogenetic Droplets. Previous work and our preliminary data suggests that by changing the affinity and valency of protein-protein interactions, both the phase behavior and properties of NBs may be tuned. However, tunable control over affinity has previously not been possible, making it impossible to construct a high resolution map of how affinity and concentration affect NB droplet formation and physical properties. Here we tackle this problem using the precise optical tunability of the Phy/PIF optogenetic system. We will express, purify, and characterize in vitro Phy/PIF repeats, which will undergo controlled phase separation as a function of light. This will lay the groundwork for rationally designing intracellular optogenetic NBs (opto-NBs). AIM 2. Build Targeted Optogenetic NBs in Cultured Cells. Having laid the foundation for systematically building tunable opto-NBs in Aim 1, we will transfer this technology to a genetically tractable cell culture system - insights into NB assembly gained from these human cell lines will have immediate human health relevance. We will examine the light-dependent assembly and properties of opto-NBs in living cells, and compare with custom theoretical models we have developed. We will extend the specificity of the technology by targeting opto-NBs using LacO arrays to nucleate droplets of tunable viscoelasticity, at defined genomic loci. AIM 3. Use Opto-NBs to Interrogate NB Assembly, Function, and Impact on Genome Architecture. We will proceed to utilize this technology to study native NBs, focusing on the nucleolus. We will measure the kinetics and viscoelastic-dependence of opto-NB recruitment of nucleolar components. Upon subsequent exposure of hybrid nucleoli/opto-NBs to pure 650nm light, they will form solid gel particles, which can then be immediately and stably extracted from lysed cells and subjected to Mass spectrometry analysis. This work will lay the groundwork for an unprecedented spatiotemporal control of perturbation and characterization of various NBs. Moreover, by optically controlling NB assembly, our approach will enable precisely controlled, time-dependent perturbations to genome architecture. This system is amenable to high-throughput assays, and we will work with other researchers in the 4D Nucleome Program to link the spatiotemporal precision of our opto-NB technology to other powerful techniques for investigating the nucleus, such as gene proximity mapping (e.g. Hi-C) [40].